cDNAs encoding HdEG66 were amplified by the polymerase chain reaction from an abalone hepatopancreas cDNA library with primers synthesized on the basis of partial amino-acid sequences of
Trang 1Purification and cDNA cloning of a cellulase from abalone
Haliotis discus hannai
Ken-ichi Suzuki, Takao Ojima and Kiyoyoshi Nishita
Laboratory of Biochemistry and Biotechnology, Graduate School of Fisheries Sciences, Hokkaido University, Japan
A cellulase [endo-b-1,4-D-glucanase (EC 3.2.1.4)] was
iso-lated from the hepatopancreas of abalone Haliotis discus
hannaiby successive chromatographies on TOYOPEARL
CM-650M, hydroxyapatite and Sephacryl S-200 HR The
molecular mass of the cellulase was estimated to be
66 000 Da by SDS/PAGE, thus the enzyme was named
HdEG66 The hydrolytic activity of HdEG66 toward
carb-oxymethylcellulose showed optimal temperature and pH at
38C and 6.3, respectively cDNAs encoding HdEG66 were
amplified by the polymerase chain reaction from an abalone
hepatopancreas cDNA library with primers synthesized on
the basis of partial amino-acid sequences of HdEG66 By
overlapping the nucleotide sequences of the cDNAs, a
sequence of 1898 bp in total was determined The coding
region of 1785 bp located at nucleotide position 56–1840
gave an amino-acid sequence of 594 residues including the
initiation methionine The N-terminal region of 14 residues
in the deduced sequence was regarded as the signal peptide as
it was absent in HdEG66 protein and showed high similarity
to the consensus sequence for signal peptides of eukaryote secretory proteins Thus, matured HdEG66 was thought to consist of 579 residues The C-terminal region of 453 residues
in HdEG66, i.e approximately the C–terminal three quar-ters of the protein, showed 42–44% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and Thermomonospora fusca While the N-terminal first quarter of HdEG66 showed 27% identity to the carbohydrate-binding module (CBM) of a Cellulomonas fimicellulase, CenA Thus, the HdEG66 was regarded as the GHF9-cellulase possessing a family II CBM in the N-ter-minal region By genomic PCR using specific primers to the 3¢-terminal coding sequences of HdEG66-cDNA, a DNA of
2186 bp including three introns was amplified This strongly suggests that the origin of HdEG66 is not from symbiotic bacteria but abalone itself
Keywords: cellulase; abalone; invertebrate; cDNA cloning; cellulase gene
Cellulase (endo-b-1,4-D-glucanase) is an enzyme which
hydrolyzes internal b-1,4-glycoside linkages of cellulose
chains [1] The cellulase has been shown to exist not only in
plants [2], molds [3], fungi [1], bacteria [1] and protista [4],
but also in herbivorous invertebrates, such as arthropods
[5–7], nematodes [8] and mollusks [9–14] Most cellulases
from microorganisms are composed of a catalytic domain
and ancillary domains such as CBMs and linkers, while the
invertebrate cellulases except for two nematode enzymes
have just a catalytic domain [1,8,14] The origin of the
invertebrate cellulases was initially explained as products of
symbiotic microorganisms in the intestine or contamination
by foods [15,16] However, those cellulases have become
considered to be the products of invertebrates themselves, as
animals bred in the presence of antibiotic could produce
cellulases [17] and cellulase genes were cloned from termite
[18,19], crayfish[20], nematode [21], and mussel [22] According to the criteria based on hydrophobic cluster analysis [23], termite and crayfishcellulases are classified into the GHF9 subfamily which includes the majority of cellulases from plants, bacteria, and a slime mold [24] Nematode cellulases are classified into GHF5 which includes some bacterial and fungal cellulases [21] On the other hand, a thermostable and low molecular mass ( 20-kDa) cellulase was recently isolated from blue mussel and the primary structure was determined [13,22] Origin of the mussel cellulase was also investigated by genomic PCR similar to the case of arthropod cellulases According to the primary structure analysis, the mussel cellulase is classified into the GHF45 subfamily 2, being distinct from the arthropod ones that are classified into GHF9 This leads us
to consider that molluscan cellulases possess somewhat different properties and a different evolutionally origin from arthropod ones However, at present there is little informa-tion about the biochemical properties and primary struc-tures of molluscan cellulases to assess the fundamental differences between molluscan and other invertebrate cellu-lases
Therefore, in the present study, we attempted to isolate a cellulase from abalone Haliotis discus hannai which is one of the most common and valuable herbivorous molluscs in Japan, and determine its primary structure In addition, we investigated the existence of a cellulase gene in abalone chromosomal DNA by genomic PCR
Correspondence to T Ojima, Laboratory of Biochemistry and
Biotechnology, Graduate School of Fisheries Sciences,
Hokkaido University, Hakodate, Hokkaido 041-8611, Japan.
Fax: + 81 138 40 8800, Tel.: +81 138 40 8591,
E-mail: ojima@fish.hokudai.ac.jp
Abbreviations: CBM, carbohydrate-binding module; GHF, glycoside
hydrolase family; CMC, carboxymethylcellulose.
Enzymes: endo-b-1,4- D -glucanase (EC 3.2.1.4).
(Received 9 October 2002, revised 3 December 2002,
accepted 20 December 2002)
Trang 2Materials and methods
Materials
Living abalones were purchased from a local market in
Hakodate, Hokkaido prefecture, Japan CMC (medium
viscosity) was purchased from ICN Bio medicals, Inc (OH,
USA), TOYOPEARL CM-650Mwas from Toyo Soda Mfg,
Co (Tokyo, Japan), Sephacryl S-200 HR was from
Amer-sham Pharmacia Biotech AAB (NJ, USA), and
Hydroxy-apatite (Fast Flow Type) was from Wako pure chemical
industries Ltd (Osaka, Japan) Lysylendopeptidase,
Oligo-tex-dT (30), TaKaRa Taq DNA polymerase, 5¢-Full RACE
and 3¢-Full RACE kits, and restriction endonucleases were
purchased from TaKaRa (Tokyo, Japan) pCR-TOPO 2.1
TA cloning kit was purchased from Invitrogen (CA, USA)
The other chemicals used were reagent grade from Wako
Pure Chemical industries Ltd (Osaka, Japan)
Determination of enzymatic activity
Cellulase activity was assayed in a 1-mL of reaction mixture
containing 0.5% CMC, 10 mMsodium phosphate (pH 7.0),
and an appropriate amount of enzyme at 30C The
reducing sugar liberated by hydrolysis of CMC was
determined by the method of Nelson and Somogyi [25]
One unit of cellulase was defined as the amount of enzyme
that liberates reducing sugars equivalent to 1.0 lmol of
glucose per min under the conditions described above
Temperature dependence of cellulase activity was assayed at
4–80C and pH 7.0 Thermal stability of cellulase was
assayed by measuring remaining activity of the enzyme that
had been incubated at 4–70C for 30 min pH dependence
of cellulase activity was assayed at 30C in reaction mixtures
adjusted at pH 3.0–9.0 with10 mMsodium phosphate
Amino-acid sequencing
The N-terminal amino-acid sequence of intact enzyme was
determined withthe sample electrically transferred to a
poly(vinylidene difluoride) membrane after SDS/PAGE
using an ABI 473 A protein sequencer (Applied Biosystems,
CA, USA) For the analysis of internal amino-acid
sequences, the enzyme was digested with 1/100 (w/w) of
lysylendopeptidase at 37C for 2 h The fragments were
separated by HPLC (LP-1000, EYLA, Tokyo, Japan)
equipped withMightysil RP-18 GP column (150· 4.6 mm)
(KANTO CHEMICAL CO., INC, Tokyo, Japan) and
subjected to the protein sequencer
SDS/PAGE
SDS/PAGE was performed with10% polyacrylamide gel
according to the method of Porzio and Pearson [26] After
the electrophoresis, the gel was stained with 0.1%
Coomas-sie Brilliant Blue R-250 in 50% methanol-10% acetic acid,
and destained with5% methanol-7% acetic acid
Zymography
Zymography for cellulase was carried out by the method of
Be´guin [27] with slight modifications as follows: The enzyme
was run on SDS/PAGE at 4C and the gel was washed with100 mL of 10 mMsodium phosphate (pH 7.0))25% 2-propanol by gently shaking at 4C for 30 min to remove SDS This washing was repeated once more and the gel was equilibrated with10 mM sodium phosphate (pH 7.0) at
4C for 30 min to accomplishrenaturation of the enzyme Then, the gel was laid on 2% agar gel (5 mm thick) containing 0.1% CMC and 10 mM Tris/HCl (pH 7.5)
whichwas solidified in Petri dish(/ 20 cm) After the incubation at 37C for 3 h, the overlaid gel was removed and the agar replica gel was stained with 0.1% Congo Red aqueous solution Location of the enzyme was detected as unstained bands
Determination of protein concentration Protein concentration was determined by the biuret method [28] or the method of Lowry et al [29] using bovine serum albumin fraction V as a standard protein
cDNA cloning Construction of the cDNA library and cloning of cellulase cDNA was achieved as follows: Total RNA was extracted from 1 g of abalone hepatopancreas by the ganidinium thiocyanate-phenol method [30] and mRNA was selected with Oligotex-dT (30) from the total RNA according to the manufacturer’s protocol Double-stranded cDNA was syn-thesized from the mRNA with a cDNA synthesis kit (TaKaRa, Tokyo, Japan) and used as an abalone cDNA library cDNAs encoding abalone cellulase were amplified
by PCR from the cDNA library with degenerated primers synthesized on the basis of partial amino-acid sequences of the cellulase PCR was carried out in a 50-lL of reaction mixture containing 50 mMKCl, 10 mMTris/HCl (pH 8.3),
2 mM eachof dATP, dTTP, dGTP and dCTP, 1.2 mM MgCl2, 2 pmolÆmL)1primers, 1 ngÆmL)1template DNA, and 0.05 unitsÆmL)1 TaKaRa Taq DNA polymerase A successive reaction at 95C for 30 s, 45 C for 60 s and
72C for 90 s was repeated for 30 cycles witha PC 700 Program Incubator (ASTEC, Fukuoka, Japan) cDNAs for 5¢-and 3¢-terminal regions of mRNA were amplified witha 5¢-Full RACE kit and a 3¢-Full RACE kit (TaKaRa, Tokyo, Japan), respectively Genomic PCR was performed withDNA primers specific to 3¢-terminal regions of the cellulase cDNA and abalone chromosomal DNA prepared from the adductor muscle by the conventional method [31] The PCR products were cloned with a pCR-TOPO 2.1 TA cloning kit (Invitrogen, CA, USA) and sequenced using a BigDye-terminator Cycle sequencing kit (Applied Biosys-tems, CA, USA) and an ABI 310 DNA sequencer (Applied Biosystems, CA, USA)
Results
Purification of cellulase from abalone hepatopancreas Hepatopancreas (125 g) dissected from 10 abalones (aver-age shell size: 8· 6 cm) were cut into small pieces with scissors and suspended in 250 mL of 10 mM sodium phosphate (pH 7.0) containing 0.2% sodium azide, 1 mM phenylmethanesulfonyl fluoride and 1 m EDTA After
Trang 3the extraction at 4C for 30 min, the extract was
centri-fuged at 10 000 g for 15 min The supernatant was applied
to a TOYOPEARL CM-650M column (2.0· 15 cm)
pre-equilibrated with10 mMsodium phosphate (pH 7.0), and
proteins adsorbed were eluted witha linear gradient of
0–200 mMNaCl in 10 mMsodium phosphate (pH 7.0) As
shown in Fig 1, three fractions showing hydrolytic activity
toward CMC, namely CM-I–III fractions, were eluted
According to SDS/PAGE followed by zymography, the
CM-I and -II were found to contain 66 000, 75 000, and
100 000 Da proteins withcellulase activity in substantial
amounts However, the CM-III fraction contained only the
66 000 Da protein in fairly high purity Therefore, in the
present study, we focused on the 66 000-Da cellulase
(named HdEG66) in the CM-III fraction and attempted
to isolate it
The CM-III fraction, i.e fractions 19–24, was applied to a
hydroxyapatite column (1.5· 20 cm) pre-equilibrated with
10 mMpotassium phosphate (pH 7.0), and adsorbed
pro-teins were eluted witha linear gradient of 10–300 mM
potassium phosphate (pH 7.0) As shown in Fig 2, the
HdEG66 was eluted as a major peak at around 0.18M
potassium phosphate, i.e fractions 25–29 As these fractions
were still contaminated by the small amount of 25 000 Da
protein, they were lyophilized and subjected to gel-filtration
through Sephacryl S-200 HR Consequently, the HdEG66
was eluted in a single major peak and showed a single band
of 66 000 Da in boththe SDS/PAGE and zymogram
(Fig 3) According to the eluting position in the
gel-filtration, molecular mass of the HdEG66 was estimated to
be 66 000 Da whichwas fairly consistent withthat
estima-ted by SDS/PAGE This indicates that HdEG66 is a
monomeric enzyme The yield and purity of the HdEG66 in
respective purification steps are summarized in Table 1 The
purified HdEG66 showed a specific activity of 13.9 UÆmg)1,
which is approximately 95-fold higher than that of crude
extract The optimal temperature and pH of the HdEG66
were at 38C and pH 6.3, respectively, and the enzyme was stable to heating at 30C for 30 min (data not shown) Partial amino-acid sequence of HdEG66
Partial amino-acid sequences of the HdEG66 were analyzed
in order to design PCR primers for the amplification of HdEG66-cDNA To analyze the N-terminal sequence, the HdEG66 was blotted onto a poly(vinylidene difluoride) membrane and subjected to the protein sequencer Conse-quently, an amino-acid sequence of 14 residues was identified as VDVTISNHWDGGFQ (Table 2) Then, in order to analyze the internal amino-acid sequences, the HdEG66 was digested withlysylendopeptidase and the
Fig 1 TOYOPEARL CM-650M column chromatography of abalone
crude extract Crude extract from abalone hepatopancreas was applied
to a TOYOPEARL CM-650M column (2.0 · 15 cm) and eluted with
a 0–0.2 M NaCl linear gradient in 10 m M sodium phosphate (pH 7.0)
at a flow rate of 30 mLÆh)1 Eachfraction contains 5.0 mL The SDS
gel electrophoretic patterns of the sample before chromatography (Cr)
and fractions indicated by the arrows a–j are shown in the inset.
M, molecular mass markers; 15 K ¼ 15 000 Da etc.
Fig 2 Purification of abalone cellulase by hydroxyapatite column chromatography The CM-III fraction in TOYOPEARL CM-650M chromatography was applied to a hydroxyapatite column (1.5 · 20 cm) and eluted witha 0.01–0.3 M potassium phosphate (pH 7.0) at a flow rate of 30 mLÆh)1 Eachfraction contains 5.0 mL The SDS gel electrophoretic patterns of fractions indicated by the arrows a–i are sh own in th e inset Th e fractions c–e were pooled.
Fig 3 Purification of abalone cellulase by Sephacryl S-200 HR gel filtration The cellulase fraction obtained in the hydroxyapatite column chromatography was concentrated by lyophilization and then applied
to a Sephacryl S-200 HR column (2.0 · 140 cm) Eachfraction con-tains 5.0 mL V 0 is the void volume of the column The SDS gel electrophoretic and zymographic patterns of fractions indicated by the arrows a–c are shown in the inset The fractions a–c were pooled as the purified HdEG66.
Trang 4fragments were isolated by HPLC Among the fragments,
LP1-LP9 fractions were subjected to sequencing (Table 2)
According to database searches on DDBJ, GenBank and
EMBL, these sequences were found to show 42–80%
identity to the amino-acid sequences of termite and
cockroachcellulases Among the fragments, LP5 and LP6
were considered to be derived from middle and C-terminal
region of the HdEG66, respectively, from the sequence
similarity to the termite cellulase Then, a forward primer F1
was synthesized on the basis of the N-terminal sequence of
the HdEG66, while reverse primers R1 and R2 were
synthesized on the basis of sequences of LP5 and LP6,
respectively (Table 2)
PCR amplification of HdEG66-cDNA
cDNAs encoding the N-terminal region of the HdEG66
were amplified by PCR using the F1–R1 primer pair This
PCR gave a cDNA withapproximately 1000 bp named
Hd1-DNA The Hd1-DNA was cloned with a TOPO
cloning kit and sequenced (Fig 4A) The amino-acid
sequence deduced from the Hd1-DNA corresponded to
the N-terminal 335 amino-acid sequence of the HdEG66
Next, in order to obtain cDNAs encoding C-terminal region
of the HdEG66, a forward specific primer F2 was newly
synthesized and PCR was performed using the F2-R2
primer pair Thus, Hd2-DNA of 477 bp encoding the
C-terminal 159 amino acids of the HdEG66 was amplified
Finally, 5¢- and 3¢-RACE PCRs were performed using primers shown in Table 2, and Hd5RACE-DNA and Hd3RACE-DNA for 5¢- and 3¢-terminal regions were amplified, respectively By combining the nucleotide sequences of the Hd5RACE-DNA, Hd1-DNA, Hd2-DNA and the Hd3RACE-Hd2-DNA in this order, the nucleo-tide sequence of total 1898 bp was determined (Fig 5) The reliability of the nucleotide sequence was confirmed with HdFull-DNA whichwas amplified withthe specific primer pair, FLF1–FLR1 (Table 2 and Figs 4 and 5) The translational initiation codon ATG was found in nucleotide positions from 56 to 58 and termination codon TAA from
1838 to 1840 (Fig 5) In the 3¢-terminal region, a putative polyadenylation signal sequence AATAAA and a poly(A+) tail were found These structural characteristics indicate that the HdEG66 cDNA is not derived from prokaryote like intestinal bacteria The translational region
of 1785 bp gave an amino-acid sequence of 594 residues All the amino-acid sequences determined with lysylendopepti-dase fragments, LP1–LP9, are found in the deduced sequence, indicating that the thus cloned cDNAs are of the HdEG66 protein It is noteworthy that the N-terminal
15 residues in the deduced sequence are absent in the HdEG66 protein According to the sequence comparison withthe consensus sequence for signal peptides of eukaryote secretory proteins [32], the N-terminal region of 14 residues except for the initiation methionine is regarded as the signal peptide of the HdEG66 Further, the inconsistent residue
Table 2 Partial amino acid sequences of HdEG66 and nucleotide sequences of primers W ¼ A/T, Y ¼ C/T, H ¼ A/C/T, H ¼ A/C/T, R ¼ A/G,
S ¼ C/G and N ¼ A/G/C/T.
Peptides Sequences Primer names DNA sequences
Intact VDVTISNHWDGGFQ F1 ACNATHWSNAAYCAYTGGGA
3RAC TTCTTCAAGGGCTGGCTCCCT
3AP CTGATCTAGAGGTACCGGATCC
5RACF ATCCTCACGAACAAGCAG
5RACR GATCGCGATGCAGGCCTT
FLF1 GGACGACTACAGCGTCTTCAGTAGA
FLR1 TCCAAACAGTCAGTTTCTTAACCGT
Table 1 Purification of cellulase from abalone Haliotis discus hannai One unit of cellulase was defined as th e amount of enzyme th at liberates reducing sugars equivalent to 1.0 lmol of glucose per min.
Purification step
Total protein (mg)
Specific activity (unitsÆmg)1)
Total activity (units)
Purification (fold)
Yield (%)
Trang 5between the deduced sequence and the sequence of LP7
peptide was found at the amino-acid position 388 Namely,
the neighboring residue of LP7 toward the N-terminus
should be lysine because LP7 is a fragment produced by
lysylendopeptidase digestion However, the corresponding
residue is not lysine but asparagine in boththe deduced
sequence and the amino-acid sequence of LP8 We now
consider that this inconsistency has arisen from
hetero-geneity of the HdEG66, e.g coexistence of proteins with
lysine and asparagine at the position 338 in the HdEG66
preparation
Amplification of HdEG66 gene from abalone
chromosomal DNA
The existence of HdEG66 gene in the abalone chromosomal
DNA was examined by genomic PCR using primers, 3RAC
and FLR1, which are specific to the 3¢-terminal region of the
HdEG66-cDNA (Table 2) By PCR, a DNA of 2186 bp
named Hdcel-1 DNA was amplified from the abalone
chromosomal DNA that was prepared from the adductor
muscle By comparison withthe sequence of
HdEG66-cDNA, the Hdcel-1 DNA was revealed to consist of three
introns (each664 bp, 757 bp, and 229 bp) and 4 exons (each
91 bp, 172 bp, 191 bp, and 82 bp) (Fig 4B) The positions
of introns in the cDNA sequence are also shown in Fig 5
The GU-AG rule in eukaryotic genes is applicable to these
intron-exon junctions These results indicate that the Hdcel-1
DNA is part of a structural gene for HdEG66 and that
HdEG66 is the product of abalone itself, and is not derived from symbiotic microorganisms, e.g intestinal bacteria
Discussion
In the present study, we have successfully isolated a cellulase HdEG66 from abalone hepatopancreas The molecular mass of HdEG66 was estimated to be 66 000 Da by both SDS/PAGE and gel-filtration through Sephacryl S-200 HR, and the optimal temperature and pH were shown to be
38C and pH 6.3, respectively In addition, the HdEG66 showed weak hydrolytic activity toward crystalline cellulose like termite cellulases (data not shown) The HdEG66 showed somewhat larger molecular size compared to the other invertebrate cellulases, however, the basic properties
of the HdEG66 were fairly similar to those of other invertebrate cellulases [6,11,12,33]
WithcDNAs amplified by PCR, an amino-acid sequence
of 594 residues for the HdEG66 was determined The N-terminal region of 15 residues including initiation methi-onine was absent in the purified HdEG66 protein and showed the characteristic feature for signal peptides of eukaryotic secretory proteins [32] Therefore, this region was regarded as the signal peptide that was cut away upon secretion of the HdEG66 Accordingly, the matured HdEG66 was considered to consist of 579 residues with the calculated molecular mass of 63 196.88 Da
By sequence comparison withother invertebrate and bacterial cellulases, the C-terminal region of 453 residues in the HdEG66 was regarded as the GHF9-type catalytic domain i.e it showed 44, 43, and 42% identity with the corresponding regions of termite [18], crayfish[20], and Thermomonospora fusca[34] cellulases, respectively (Fig 6) Further, the catalytically important residues in GHF9 cellulases [35–38], i.e His506, Asp200, Asp203, Asp550 and Glu559 in the HdEG66 sequence were all conserved (Fig 6) Based on these results, we conclude that the HdEG66 is classified into GHF9 On the other hand, HdEG66 was found to possess an extended N-terminal region of 126 residues which is deficient in other invertebrate cellulases (Fig 6) This extended region showed sequence identity of 27% with the CBM attached by a linker in Cellulomonas fimi CenA [39] The CBM of CenA belongs
to CBM family II, which is currently the largest among five principal families, i.e families I–IV and VI [1] The family II CBMs possess strictly conserved four tryptophans and highly conserved two cysteines that form a disulfide bridge
In case of N-terminal extended region of the HdEG66, three out of the four tryptophans are conserved at residues 24, 43, and 79, although the remaining one is substituted by aspartic acid at residue 57 While the two cycteines are not conserved in HdEG66, three cysteines are however present
at residues 33, 58, and 90 In addition, a putative linker region richin threonine and glycine residues locates in the position connecting the N-terminal extended region and the catalytic domain (Fig 6) These sequence charac-teristics strongly suggest that the N-terminal extended region of the HdEG66 corresponds to a family II CBM followed by a linker Accordingly, HdEG66 is considered
to be the first animal cellulase possessing the family II CBM
in the N-terminus of the GHF9-type catalytic domain Cellulose-binding ability and other biochemical functions
Fig 4 Structures of cDNA and genomic fragment for HdEG66 (A)
structure of HdEG66 cDNA Open and closed boxes indicate
trans-lational and untranstrans-lational regions, respectively Relative positions of
Hd1-DNA, Hd2-DNA, Hd5RACE-DNA, Hd3RACE-DNA, and
HdFull-DNA are indicated as solid lines Bold lines in bothsides of the
cDNAs indicate primers used for th e PCR Length s of th e cDNAs are
shown in the parentheses (B) Structure of the genomic fragment,
Hdcel-1 DNA Open and closed boxes represent exons and introns,
respectively The sequence data for HdEG66 cDNA and Hdcel-1
DNA are available from DNA Data Bank of Japan withaccession
numbers, AB092978 and AB092979, respectively.
Trang 6for the putative CBM of HdEG66 are now under
investi-gation
By PCR withchromosomal DNA prepared from abalone
adductor muscle and specific primers to the 3¢-region of
HdEG66-cDNA, a genomic fragment Hdcel-1 DNA
enco-ding for C-terminal region of the HdEG66 was amplified The Hdcel-1 DNA consisted of four exons and three introns (Fig 4B) and the coding sequence of the exons was consistent with that of cDNA This strongly suggests that the Hdcel-1 DNA is a genomic fragment derived from
Fig 5 The nucleotide and deduced amino-acid sequences of the HdEG66 Residue numbers for bothnucleotide and amino acid are indicated in the right of each row The translational start codon ATG, termination codon TAA, and a putative polyadenylation signal AATAAA are boxed A putative signal peptide is indicated by a dotted underline The amino-acid sequences determined with intact HdEG66 (N-terminus) and peptides LP1–LP9 are indicated by lines under the amino-acid sequence The positions of primers for PCR are indicated by lines above the nucleotide sequence The Asp338 that was suggested to be lysine from the LP7 sequence was double-boxed Introns 1–3 indicates the positions of introns revealed by the analysis of a genomic fragment Hdcel-1 DNA (see Fig 4B).
Trang 7abalone chromosome not from intestinal symbiotes and that
the HdEG66 is an enzyme secreted by abalone itself Further,
the position of intron-2 in the Hdcel-1 DNA was found to
coincide to the position of corresponding intron in termite
cellulase gene [19] These results strongly suggest that the
HdEG66 and termite cellulase genes derive from a common
ancestral gene although the termite cellulase lacks CBM
In the present study, we found the presence of cellulases
of approx 75 000 and 100 000 Da as well as HdEG66 (see
Fig 1) We are now attempting to purify these isoforms and
determine their primary structures in order to reveal the
structural characteristics of abalone cellulases and their
evolutionary relationships to other invertebrate and
bacter-ial cellulases
References
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5 Watanabe, H., Nakamura, M., Tokuda, G., Yamaoka, I., Scriv-ener, A.M & Noda, H (1997) Site of secretion and properties of endogenous endo-b-1,4-glucanase components from Reticulitermes speratus (Kolbe), a Japanese subterranean termite Insect Biochem Mol Biol 27, 305–313.
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7 Xue, X.M., Anderson, A.J., Richardson, N.A., Anderson, A.J., Xue, G.P & Mather, P.B (1999) Characterisation of cellulase
Fig 6 Comparison of amino-acid sequences
for the HdEG66 and other cellulases The
sequence of C-terminal 453 residues for the
HdEG66 was aligned withthe sequences for
catalytic domains of RsEG (Reticulitermes
speratus [18]), CqEG (Cherax quadricarinatus
[20]), and E4-68 (Thermomonospora fusca
[37]) Residue numbers of RsEG, CqEG, and
E4-68 are of the precursor enzymes
Catalyti-cally important residues in GHF9 are boxed.
Identical residues among the sequences are
indicated by asterisks While the sequence of
N-terminal 126 residues except for the
puta-tive signal peptide region was aligned withthe
CBM attached by a linker of Cellulomonas
fimi CenA [39] Locations of four tryptophans
strictly conserved in family II CBMs [1] are
indicated by reverse triangles.
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